Journal: European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery

Article Title: Decellularized mitral valve in a long-term sheep model.

doi: 10.1093/ejcts/ezx485

Figure Lengend Snippet: Figure 7: Immunostaining of the decellularized mitral valve (DMV) and the biological mitral valve (BMV). (A and B) DMV and (C and D) BMV. (A, C) CD45 and DNA; (B, D) procollagen I and DNA. DMV shows collagen-producing cells within the leaflets. BMV has almost no cells, positive for procollagen I. The CD45-positive cells in the BMV tend to form nodules (picture of the region, close to the annulus). BMV has only several CD45-positive cells on the surface. Ao: Aorta ascendens.

Article Snippet: D ow nloaded from https://academ ic.oup.com /ejcts/article/53/6/1165/4828177 by guest on 22 M ay 2024 • Collagen IV (clone CI22, DAKO, Hamburg, Germany) • Collagen I C2345 clone COL-I (Sigma-Aldrich Chemie GmbH, Munich, Germany) • CD31 [monoclonal mouse immunoglobulin G (IgG)2a, Serotec, Raleigh, NC, USA] • von Willebrand factor (polyclonal rabbit IgG, DAKO) • Endothelial nitric oxide synthase (monoclonal mouse IgG1, eNOS/NOS Type III, BD Transduction Laboratories, San Jose, CA, USA) • CD45 [mouse monoclonal (clone OX-1) IgG, BioRad, Oxford, UK] • Procollagen (monoclonal mouse IgG1, M-38-c, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) The images were obtained using the microscope AxioObserver A1, with the camera AxioCamMRm (Carl Zeiss, Jena, Germany).

Techniques: Immunostaining

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a Detection of endogenous hTERT. The cells were treated with DMSO (denoted as “non M”) or nocodazole to manipulate cells in mitosis (denoted as “M”), and immunoprecipitated with anti-hTERT mouse mAb (10E9-2) or anti-hTERT sheep pAbs (abx120550). The hTERT proteins were detected by anti-hTERT mouse mAb (2E4-2) (two left panels) or anti-hTERT rabbit mAb (ab32020) (two right panels). b hTERT proteins were isolated by immunoprecipitation with 10E9-2 and detected by 2E4-2 (upper panel). Cells arrested in mitosis with nocodazole were confirmed by anti-phospho-histone H3 (Ser10) antibodies (lower panel). c Endogenous hTERT immunoprecipitated from 293T cells were treated with λ phosphatase to remove the phosphate groups and detected by 2E4-2 (upper panel). d Summary of MS analysis to identify phosphorylation sites in hTERT. The numbers of phosphopeptides and nonphosphopeptides are based on pep_expect (Expectation value for PSM) < 0.05 used as a cutoff value. Bold letters indicate amino-acid residues identified as phosphorylated residues. See the details in Supplementary Data . e Multiple sequence alignment of telomerases using COBALT . Red letters denote conserved residues and blue letters denote non-conserved residues with no gaps. Yellow is the phosphorylated threonine residue detected in this study. f The immune complexes were treated with λ phosphatase. Phosphorylation of hTERT at T249 was detected by anti-249T-P rabbit pAbs. g Endogenous hTERT proteins from cells transfected with two different siRNAs specific for hTERT . Phosphorylation of hTERT at T249 was detected by anti-249T-P pAbs (upper panel). Whole-hTERT proteins were detected by 2E4-2 (lower panel). h The recombinant hTERT fragment proteins (191–306 a.a) were phosphorylated by CDK1-cyclinB or IKK2_2-664 in vitro and phosphorylated proteins were detected in Phos-Tag SDS-PAGE (upper panel). Phosphorylation of hTERT at threonine 249 by CDK1-cyclinB was confirmed by anti-249T-P pAbs (lower panel). The protein bands enclosed with square were analyzed by MS to confirm the phosphorylation sites. i Phosphorylation of threonine 249 by CDK1-cyclinB was confirmed by anti-249T-P pAbs and TpMab-1 mouse mAb. Experiments were repeated three times (for a , g , i ), five times (for b , c , f ) and twice (for h ) with similar results. Source data are provided in the Source Data file.

Article Snippet: Anti-hTERT mouse mAb (1:100, clone 2E4-2), anti-hTERT rabbit mAb (1:1000, Abcam, ab32020), anti-249T-P pAbs (1:1000), TpMab-1 mAb (1:50), anti-phospho-histone H3 (Ser10) rabbit pAbs (1:2000, Merck, 06-570), anti-cdc2 [CDK1] (POH1) mouse mAb (1:1000, Cell Signaling Technology, 9116), anti-β-actin (AC-15) mouse mAb (1:20000, Sigma-Aldrich, A5441), anti-FOXO4 rabbit mAb (1:4000, Abcam, ab128908), anti-GAPDH mouse mAb (1:1000, MBL Co., Ltd., M171-3), and anti-SMG6 rabbit pAbs (1:1000) were used for immunoblotting (IB).

Techniques: Immunoprecipitation, Isolation, Phospho-proteomics, Sequencing, Residue, Transfection, Recombinant, In Vitro, SDS Page

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a – e Representative images of precancerous lesions stained with anti-249T-P pAbs. N-small, intercalated duct to intralobular duct; N-large, interlobular duct to main pancreatic duct; PanIN, pancreatic intraepithelial neoplasia; Cancer, pancreatic invasive ductal adenocarcinoma. Original magnification x400, scale bar: 50 µm. f Phosphorylation of hTERT T249 was detected in the nucleus of pancreatic adenocarcinomas. Original magnification x400, scale bar: 50 µm. g , h Both normal mitotic (pointed by arrows, g ) and atypical multipolar mitotic images (pointed by arrow, h ) exhibited phosphorylation of hTERT T249 (inserts). Original magnification x400. i Expression of CDK1 was detected in the nucleus of pancreatic adenocarcinomas. Original magnification x400, scale bar: 50 µm. j Relationship between pancreatic ductal lesions and hTERT phosphorylation at T249. Box-and-whisker plot of hTERT phosphorylation for each of the pancreatic ductal lesions surgically resected from patients with pancreatic cancer ( n = 47). The box portion of the box plot is defined by two lines at the 25th percentile and 75th percentile. A line is drawn inside the box at the median. The two whisker boundaries are the 5th percentile and 95th percentile. p < 0.05 by Speaman’s rank correlation test. k – p IHC staining with anti-249T-P pAbs in non-cancerous lesions ( k , l ) and HCC lesions ( m , n ) obtained from two individual patients. IHC staining with anti-CDK1 pAbs in HCC lesions ( o , p ) obtained from two identical patients. Original magnification x200, scale bar: 100 µm. q , r Kaplan–Meier survival analysis of primary patients with HCC grouped for 249T-P-positive (red) or negative (blue). Phosphorylation status of hTERT T249 ( q ) and CDK1 expression ( r ) showed statistically significant correlations with overall survival (OS, p = 0.048 and 0.036, respectively, log rank test, two-sided) in patients.

Article Snippet: Anti-hTERT mouse mAb (1:100, clone 2E4-2), anti-hTERT rabbit mAb (1:1000, Abcam, ab32020), anti-249T-P pAbs (1:1000), TpMab-1 mAb (1:50), anti-phospho-histone H3 (Ser10) rabbit pAbs (1:2000, Merck, 06-570), anti-cdc2 [CDK1] (POH1) mouse mAb (1:1000, Cell Signaling Technology, 9116), anti-β-actin (AC-15) mouse mAb (1:20000, Sigma-Aldrich, A5441), anti-FOXO4 rabbit mAb (1:4000, Abcam, ab128908), anti-GAPDH mouse mAb (1:1000, MBL Co., Ltd., M171-3), and anti-SMG6 rabbit pAbs (1:1000) were used for immunoblotting (IB).

Techniques: Staining, Phospho-proteomics, Expressing, Whisker Assay, Immunohistochemistry